This project will analyze the function of the LcrE protein in Yersinia pestis. LcrE is implicated in the regulation of a unique secretion mechanism for virulence proteins, the Yop proteins of Yersinia. The Yops lack identifiable signal sequences and are secreted without proteolytic processing. Expression and secretion of Yops in vitro regulated by calcium. Eucaryotic cells triggers the translocation of at least one Yop directly from the surface attached bacterium into the host cell. This appears to be a mechanism of broad interest since it appears to be present in other pathogens as well. This application proposes to identify functional domains of LcrE required to target LcrE to the Yop secretion apparatus, identify domains required to regulate Yop secretion, and identify proteins that interact with LcrE. This will be accomplished by deletion analysis of LcrE, characterization of second- site mutations to identify proteins that interact with LcrE. Protein affinity chromatography, affinity blotting and immunoprecipitation will be used to confirm these interactions. Finally, the effect of LcrE mutants on the translocation of YopE into eucaryotic cells will be determined. These experiments are designed to map regions and residues of LcrE to specific functions and reveal how LcrE functions.